A biosignature based on long-term plasma protein changes specifically associated with chronic drug exposure can be used to identify patients predisposed to repeated drug use and thereby facilitate early intervention. We will test the feasibility of finding such a signature using chronic exposure of rats to addictive drugs and differential proteomic expression. Rats will receive treatment with cocaine or a non-addictive dopamine transporter inhibitor, or morphine, or saline according to a 14 day escalating dose schedule (chronic exposure) and plasma collected up to 75 days following last drug treatment. Proteomic analysis will involve stepwise depletion of abundant plasma proteins followed by liquid chromatography coupled online with mass spectrometry to analyze both the depleted proteome consisting of very low abundance proteins and the bound proteins of intermediate abundance. Proteins whose altered expression is maintained for several weeks, e.g. 30 days, and eventually return to baseline would be candidate biomarkers that could be used to detect recent exposure at a time when the addictive drug can no longer be readily measured. Top candidate will be validated by multiple reaction monitoring in a quantitative multiplexed assay that does not require antibodies to the proteins. Proteomic changes will be examined for temporal correlation with behavioral and neurochemical sequelae of chronic drug treatment that will be measured in parallel. Statistical analysis and data mining will be used to identify a biosignature with the desired properties. A successful feasibility study would stimulate and inform similar studies on human subject samples. PUBLIC HEALTH RELEVANCE: There is an unmet need for analytical biomarkers, a biosignature, of chronic drug exposure that would help to identify patients predisposed to repeated drug. An effective test could become part of routine medical care that would identify patients in need of treatment for substance use disorders and mitigate the personal and societal burdens of drug addiction.